Journal of Experimental Botany, Vol. 71, No. 2 pp.

642–652, 2020
doi:10.1093/jxb/erz150  Advance Access Publication April 13, 2019
This paper is available online free of all access charges (see https://academic.oup.com/jxb/pages/openaccess for further details)

RESEARCH PAPER

The importance of slow canopy wilting in drought tolerance

in soybean
Heng Ye1, Li Song1,2, William T. Schapaugh3, Md Liakat Ali4, Thomas R. Sinclair5, Mandeep K. Riar5,
Raymond N. Mutava1, Yang Li1, Tri Vuong1, Babu Valliyodan1, , Antonio Pizolato Neto1, Mariola Klepadlo1,
Qijian Song6, J. Grover Shannon4, Pengyin Chen4 and Henry T. Nguyen1,*,

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1 
Division of Plant Sciences, University of Missouri, Columbia, MO 65211, USA
2 
Joint International Research Laboratory of Agriculture and Agri-Product Safety, Institutes of Agricultural Science and Technology
Development, Yangzhou, 225009, China
3 
Agronomy Department, Kansas State University Manhattan, KS 66506, USA
4 
Division of Plant Sciences, University of Missouri-Fisher Delta Research Center, Portageville, MO 63873, USA
5 
Department of Crop Science, North Carolina State University, Raleigh, NC 27695, USA
6 
Soybean Genomics and Improvement Laboratory, USDA-ARS, Beltsville, MD 20705, USA

*Correspondence: [email protected]

Received 19 December 2018; Editorial decision 19 March 2019; Accepted 8 April 2019

Editor: Christine Foyer, University of Birmingham, UK

Abstract
Slow canopy wilting (SW) is a water conservation trait controlled by quantitative trait loci (QTLs) in late maturity group
soybeans [Glycine max (L.) Merr.]. Recently, two exotic (landraces) plant introductions (PI 567690 and PI 567731) were
identified as new SW lines in early maturity groups. Here, we show that the two PIs share the same water conserva-
tion strategy of limited maximum transpiration rates as PI 416937. However, in contrast to PI 416937, the transpiration
rates of these PIs were sensitive to an aquaporin inhibitor, indicating an independence between limited maximum
transpiration and the lack of silver-sensitive aquaporins. Yield tests of selected recombinant inbred lines from two
elite/exotic crosses provide direct evidence to support the benefit of SW in drought tolerance. Four SW QTLs mapped
in a Pana×PI 567690 cross at multiple environments were found to be co-located with previous reports. Moreover, two
new SW QTLs were mapped on chromosomes 6 and 10 from a Magellan×PI 567731 cross. These two QTLs explain the
observed relatively large contributions of 20–30% and were confirmed in a near-isogenic background. These findings
demonstrate the importance of SW in yield protection under drought and provide genetic resources for improving
drought tolerance in early maturity group soybeans.

Keywords:   Drought, QTL mapping, slow canopy wilting, soybean, transpiration, water use efficiency.

Introduction
Climate changes increase the occurrence of extreme weather yield losses of major crops keep increasing, despite the pro-
patterns, including irregular rainfall patterns, higher tempera- gressive increase in yield through breeding and management
ture, and the consequent drought stress, which cause signifi- practices since the 1960s (Boyer, 2013; Lobell et  al., 2014;
cant reductions in crop production (Lesk et al., 2016). These Rosenzweig et al., 2014). A decline of 30–82% in crop yields

© Society for Experimental Biology 2019.

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 Slow canopy wilting improves drought tolerance in soybean  |  643

has been projected by the end of the century under the current the SW trait in soybean. In addition, other mechanisms, such as
warming scenarios (Hatfield et al., 2011). more water resource exploration by roots, could also result in
Soybean has been estimated to have an ~40% reduction the SW phenotype. PI 416937 was found to have a much larger
in yield as a result of drought (Specht et al., 1999). To over- root system in the field than other FW lines, which might
come the negative impacts of drought stress in soybean, many be one factor to enhance the SW phenotype under drought
strategies have been developed and adopted, mainly including (Hudak and Patterson, 1995).
agricultural practices and genetic improvement of soybean Many quantitative trait locus (QTL) mapping efforts have
cultivars (Turner, 2000). Rainfall and irrigation water are been conducted to identify genomic regions associated with
being used more efficiently; however, adaptation of irriga- the SW trait (Valliyodan et  al., 2017). Initial QTL mapping
tion systems is region limited and would substantially increase work was conducted in a recombinant inbred line (RIL)
the costs of soybean production (Kebede et al., 2014). Clearly, population derived from the Benning and PI 416937 cross.
improving drought tolerance of soybean varieties is strategic- Seven QTLs were mapped in that population and the one
ally crucial to protect yield gain (Devi et  al., 2014; Ye et  al., on chromosome (Chr.) 12 was identified as a major QTL
2018a). Drought tolerance in soybean should be translated across all five independent environments with a phenotypic

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to traits enhancing yield stability rather than that increasing contribution up to 27% (Abdel-Haleem et  al., 2012). In an-
survivability under drought (Bartels et al., 2006; Blum, 2009; other QTL mapping study conducted using five populations
Passioura, 2010; Sinclair, 2011; Ye et al., 2018a). The essential under 15 environments (site or year), a total of 13 QTLs were
factor to improve drought tolerance in crops, including soy- mapped for canopy wilting with varied phenotypic contribu-
bean, is to use available water more effectively through the tions (R2=4–29%), and the majority of these QTLs (11/13)
growing season so that the plant’s physiological activity is sus- had donor alleles for SW from either PI 416937 or Jackson
tained through to maturity (Turner et al., 2001; Blum, 2009; (Hwang et al., 2015). Among these 13 QTLs, eight were further
Sinclair, 2018;Ye et al., 2018a). confirmed and their confidence intervals were refined through
Soybean genotypes differ in the time of onset and the se- Meta-QTL analysis (Hwang et al., 2016). A genome-wide as-
verity of canopy wilting in response to drought (Sloane et al., sociation study (GWAS) was also conducted, and 23 putative
1990; Carter et al., 1999; Valliyodan et al., 2017). As soil dries, loci were identified as being associated with canopy wilting
soybean genotypes with a slow canopy wilting (SW) pheno- from a diverse panel of 373 MG IV soybean genotypes (Kaler
type have delayed canopy/leaf wilting when compared with et al., 2017), indicating the complexity of the canopy wilting
fast canopy wilting (FW) lines (Charlson et al., 2009;Valliyodan trait. Although statistically significant QTLs for SW have been
et al., 2017). Field evaluations of plant introductions (PIs) from reported in soybean, most were found to be unstable across
the USDA Soybean Germplasm Collection identified PI independent environments and populations. For instance, the
416937 [maturity group (MG) VI] and PI 471938 (MG V)] as major QTL on Chr. 12 was not detectable in any populations
often expressing the SW phenotype relative to commercially or environments reported by Hwang et al. (2015, 2016); thus
available cultivars (Carter et  al., 2006; Charlson et  al., 2009). QTL confirmation needs to be performed to validate each
This trait was predicted using a simulation model to improve individual QTL, especially in a near-isogenic background. In
yield under drought by >80% of the growing seasons in most addition, effort was put into QTL mapping for response of
regions of the USA (Sinclair et  al., 2010). Currently in the transpiration to silver ions in the Benning×PI 416937 popu-
USA, the SW phenotype has been used as one of the indica- lation. Four QTLs were identified (R2=17.7–24.7%), with the
tors to screen drought tolerance in the field (Charlson et al., largest one located on Chr. 12 (Carpentieri-Pipolo et al., 2012).
2009) and several drought-tolerant soybean varieties for late However, the donor allele at this major locus for insensitivity
MGs (V–VIII) have been developed by utilizing these two PIs, of transpiration to silver ions is from an FW parent, Benning
PI 416937 and PI 471938 (Devi et al., 2014). (Carpentieri-Pipolo et al., 2012). This result is contradictory to
One of the physiological bases for SW in PI 416937 was the QTL mapping for SW in the same population, as the donor
found to be associated with limited transpiration rate (TR) allele at this locus for SW is from the SW parent, PI 416937
under atmospheric vapor pressure deficits (VPDs) of ~2.1 kPa (Abdel-Haleem et al., 2012).Thus, the correlation between the
or higher (Fletcher et  al., 2007). This limitation on TR was lack of silver-sensitive aquaporins and the SW phenotype in-
shown to be associated with limited hydraulic conductance be- volved in a limited maximum TR needs to be reconsidered.
tween the xylem and the guard cells in the leaves (Sinclair et al., Recently, two drought-tolerant exotic (unadapted) soybean
2008). This hydraulic restriction was suggested to be associated germplasms, PI 567690 and PI 567731 (MG III), were identi-
with lack of a specific silver-sensitive population of aquaporins, fied to express the SW phenotype consistently in the field com-
as the TR in PI 416937 was insensitive to AgNO3 treatment pared with two drought-sensitive cultivars, Pana and Magellan
(Sadok and Sinclair, 2010). Interestingly, response in transpir- (Pathan et al., 2014).The early maturity group of these two PIs
ation to increasing VPD of the other SW line, PI 471938, was favors their use in breeding efforts for higher latitude regions.
found to be linear without a breakpoint. Furthermore, tran- However, the mechanisms underlying the SW trait of these PIs
spiration in PI 471938 was sensitive to the AgNO3 treatment, have not yet been studied. Moreover, the impacts of SW trait
indicating the presence of an AgNO3-sensitive pathway in on yield protection under drought obviously require further
their leaves allowing higher hydraulic conductance (Sadok and investigation. The objectives of this study were (i) to elucidate
Sinclair, 2010). These results indicate that there are at least two the physiological and genetic mechanisms involved in the SW
distinct water conservation mechanisms that are involved in trait in these two germplasm sources; (ii) to identify the effects
644 | Ye et al.

of SW on yield protection under drought stress in similar gen- A

etic backgrounds; and (iii) to confirm selected major genetic
factors (QTLs) regulating SW.

Materials and methods

Plant materials
Two mapping populations were developed from soybean Pana×PI 567690 1 3 5
and Magellan×PI 567731 crosses using a single-seed descent method to
produce 258 and 160 F7:8-derived RILs, respectively (see Supplementary
Fig. S1 at JXB online). The RILs were further advanced to increase seeds B
at the Delta Research Center (DC), University of Missouri, Portageville, 4.0
PI 567731 Magellan R2(G) = 0.57

Canopy wilting score

MO, USA. Forty-six RILs from each of these populations were selected
for yield test based on contrasting canopy wilting and other agronomic PI 567690 Pana
3.0

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traits, including plant height, lodging, and maturity (Supplementary Table
S1). Near-isogenic lines (NILs) of the major QTLs were identified from
heterogeneous inbred families at the F7:8 generation (Supplementary Fig. 2.0
S1). A single F7:8 plant, which has heterozygous genotypes at the QTL
regions, was selected from the RIL families as described by Ye et al. (2015,
2018b). The NILs were selected in the progeny population of this F7:8 1.0
plant (Supplementary Fig. S1).

0.0
Plant cultivation and SW evaluation in the field
The parental lines and RIL populations were evaluated for the SW trait at
Salina BREC Delta Center
the Bradford Research and Education Center (BREC), Columbia, MO,
the DC, and Salina, KS, USA in 2013, 2014, and 2015. In Salina, KS, C 25
10 cm
Soil moisture (w/w) %
these RILs were planted in plots 3 m long and 0.75 m row space with
60 seeds per row. In Missouri, hill plots were planted with 0.75 m be- Irrigated
20 40 cm
tween rows and 0.75 m between hills. Ten seeds were sown per hill and,
3 weeks after germination, plants were thinned to four plants per hill.
Hill plot was previously approved as an effective plot design to evaluate 15 Drought
the SW phenotype, as the designed plant density (four plants per hill)
can result in enough competition for water imposing stress on the plants 10
under rain-fed conditions (Sloane et  al., 1990). To develop NILs, seeds
were sown in plots 3 m long and 0.75 m row space with 60 seeds per row 5
at BREC. At the DC, water available to the plants was monitored using
a Delta-T moisture PR2 probe (Delta-T instruments, Cambridge, UK) 0
at depths of 10 cm and 40 cm. In all the fields, scoring for leaf wilting
started when the plants were from R2 to R5 growth stages, when the
canopy was fully developed (Fehr et al., 1971; Pathan et al., 2014). Canopy
wilt scoring (WS) was rated when any of the FW check (Pana) showed
obvious canopy wilting at late morning or mid-day. Data were recorded
for leaf wilting using a 1–5 scale (1=no wilting, 2=few top leaves showed
wilting, 3=half of the leaves showed wilting, 4=severe wilting, ~75% of Fig. 1.  Canopy wilting and water use of the parental lines in the field.
the leaves showed wilting, and 5=severely wilted) (Fig. 1A). (A) Field phenotyping for canopy wilting using the canopy wilting score
In data analysis, the model for the WS trait was: (WS). WSs were used to represent canopy wilting levels from 1 to 5. (B)
Canopy wilting of the parental lines under drought conditions. Data were
yijmk = µ + gi + lj + (gl)ij + (gt)im + (glt)ijm + bk ( j) = eijmk (1) recorded for the lines at three locations for 2–3 years. Significant genotypic
differences were observed with P<0.0001, explaining 57% of phenotypic
 variations based on ANOVA across the three locations. (C) Soil moisture
where µ is the grand mean, gi is the genetic effect of the ith genotype, underneath the parental lines. Soil moisture was measured at 10 cm
lj is the effect of the jth location, tm is the effect of the mth year, (gl)ij is and 40 cm underneath the soil surface of the parental lines planted in
the interaction between the ith genotype and the jth location, (gt)im is the sandy soil at Delta Center, MO in 2014. Measurements were taken 3
the interaction between the ith genotype and the mth year, (glt)ijm is the weeks after the last rain. Columns and bars represent the mean and SDs
interaction among the ith genotype, the jth location, and the mth year, of 10 replicates.
bk(j) is the block (kth) effect within the jth location, and eijmk is a random
error following N (0, σ2e). Estimation of variance components was per-
formed by the GLM procedure in SAS version 9.3 (SAS Institute, Inc., Once four fully expanded leaves had been attained, plants were placed
Cary, NC, USA). in individual chambers, in which VPD was varied. Three VPD exposures
on two consecutive days were imposed in the following sequence: low
(0.5–1.5 kPa), medium (1.5–2.5 kPa), and high (2.5 and higher kPa). The
Evaluation of transpiration responses to VPD and silver nitrate TR for the plant was expressed as water loss rate per unit of leaf area.
Transpiration responses of the four parental lines (Pana, Magellan, PI The TR for each genotype was regressed against the VPD for each
567690, and PI 567731)  were determined as previously described genotype. For statistical analysis, two regression equations were ap-
(Fletcher et al., 2007). Plants were grown for 4 weeks at 30/26 °C day/ plied using least-squares regression in Graphpad prism 2.01 (Graphpad
night temperature in a greenhouse at the North Carolina State University. Software Inc., San Diego, CA, USA). A  key output of the regression
 Slow canopy wilting improves drought tolerance in soybean  |  645

analysis was the VPD value for the breakpoint (BP) where the two linear Results
segments intersected.
Canopy wilting and water usage of parental lines in
If VPD < X0 , TR + S1 (VPD) + C1 (line1) the field
The four parental lines were planted at three locations over
If VPD ≥ X0 , TR + S2 (VPD) + C2 (line2)
(2) multiple years to evaluate canopy WSs (Fig. 1A). The two
where X0 is the BP between the two line-segments, S1 and S2 the slopes exotic parents, PI 567731 and PI 567690, showed almost
of the first and second line segments, respectively, and C1 and C2 are the no canopy wilting (WS <1.2) compared with the two elite
constants of the first and second line segments, respectively. The slopes parents, Magellan and Pana, across all of the three locations of
of the two linear regressions (S1 and S2) were statistically compared to both hill pots and row plots (Fig. 1B). Significant genotypic
determine if they differed significantly (P<0.05). If the slopes differed,
the double-linear regression was retained. When the slopes were not sig-
differences in WS were identified between the exotic and elite
nificantly different, a simple linear regression was applied to all the data parents, explaining 57% of phenotypic variances across envir-
for that genotype. onments (locations and years) (Fig. 1B). Under the irrigation
Silver nitrate (AgNO3) treatment and subsequent data analysis were condition (non-stress), the FW line showed significantly less

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performed as previously outlined (Sadok and Sinclair, 2010). Plants were soil moisture at 10 cm and 40 cm depth soil layers, compared
grown for 4 weeks in the greenhouse at a 30/26  °C day/night tem-
perature scheme. The shoots were detached 4 weeks after emergence.
with the two SW lines (Fig. 1C). Under the rain-fed condition
The subsequent change in transpiration was measured gravimetrically. (drought), the surface soil moistures (10 cm depth) were all de-
The VPD of the growth chamber was kept in the range of 3.3–4.0 kPa. creased to a level of ~5% (w/w); however, at deeper soil layers
The percentage reduction in transpiration rate (RTR) for each individual (40 cm depth), the two SW lines retained >12% moisture con-
plant as a result of the inhibitor treatment was calculated using the fol- tent compared with 7% by the FW line, Pana (Fig. 1C). These
lowing equation for 10 plants for each line.
results suggest that the SW phenotype in these exotic lines is
RTR = 100 × (TR0 TRx ) /TR0
(3) involved in water use efficiency and conservation.

where TR0=plant transpiration rate in water and TRX=plant transpir-

ation rate in AgNO3 solution. Transpiration responses to VPD and an aquaporin inhibitor
The responses of transpiration in the four parental lines to
Yield tests under irrigated and rain-fed field conditions increasing VPD were further evaluated. Among these four lines,
Standard four row yield plots were planted to evaluate yield of the selected PI 567690, PI 567731, and Magellan were found to exhibit a
RILs in the sandy soil at two locations: Salina, KS, and the DC, MO. At BP in their TRs as VPD was increased (Fig. 2A), similar to PI
each location, two sets of yield plots were planted with three replications,
subjected to two treatments (irrigated and rain-fed). For each plot, 600
416937 (Fletcher et  al. 2007). PI 567690 exhibited a BP in
seeds were sown in 4 m rows with 0.75 m row space. Only the two center TR at 2.00 kPa, while PI 567731 and Magellan exhibited BPs
rows were harvested for yield evaluation. Drip irrigation was applied to at lower VPDs of 1.38 and 1.46 kPa, respectively (Fig. 2A). It
plots once in 10 d without rainfall in July and August for irrigated treat- was noted that PI 567690 showed the strongest suppression in
ments. Four and two irrigations were applied at Salina, KS, and the DC, transpiration when VPD was increased (Fig. 2A). The FS line,
MO, respectively. No irrigation was applied for rain-fed treatments at
both locations. Rain-fed conditions were considered as relatively drought
Pana, did not exhibit a BP, and a single linear regression was
conditions compared with the irrigated conditions, especially the poor fit (non-BP line) (Fig. 2A). These results showed that the three
water-holding capacity of sandy soils. lines with BPs in TRs (BP lines) had limited maximum TRs
and could be candidate donors for SW (Fletcher et al., 2007).
Linkage map construction and QTL analysis
Detached shoots were treated with an aquaporin inhibitor
(AgNO3) and significant RTRs were observed in the all four
Genomic DNA of the parents and the RILs of the two RIL popula-
tions was extracted using a standard cetyltrimethylammonium bromide parental lines (Fig. 2B).The three BP lines (PI 567690, PI 567731,
(CTAB) method (Doyle and Doyle, 1990) with minor modifications. and Magellan) showed greater sensitivities in transpiration to
Single nucleotide polymorphism (SNP) genotyping was performed using the AgNO3 treatment than the non-BP line (Pana), unlike the
the BARCSoySNP6K Illumina Infinium BeadChips (Illumina Inc., San nearly complete lack of sensitivity in transpiration to silver ions
Diego, CA, USA) at the Washington University genotyping facility, St. found previously in PI 416937 (Sadok and Sinclair, 2010).These
Louis, MO, USA. The SNP alleles were called using the GenomeStudio
Genotyping Module (Song et al., 2013). A genetic linkage map of each results indicate that the limited transpiration imposed in the
mapping population was constructed using the program JoinMap 3.0 BP lines could be independent of the lack of a silver-sensitive
(van Ooijen and Voorrips, 2001). A LOD (logarithm of odds) score of 3.0 aquaporin population hypothesized for PI 416937. In future, the
was used for two-point analysis and a LOD score of 2.0 was used for all focus should be on all aquaporin populations including both
three-point and multipoint analysis. Putative QTLs for the traits studied silver-sensitive and -insensitive aquaporin to understand the
were initially detected by the interval mapping (IM) method using the
program MapQTL 5.0 (van Ooijen, 2004). Composite interval mapping regulation of limited transpiration under high VPD.
(CIM) was then performed using the multi-QTL method and the ap-
propriate cofactors (van Ooijen and Voorrips, 2001). A LOD score sig-
nificance threshold value was estimated for each trait in each location by Inheritance of SW and its effects on yield protection
1000 permutations to determine a QTL at the genome-wide significance under drought
level of P=0.05 (Doerge and Churchill, 1996).The name of the identified
QTL was nominated following SoyBase nomenclature requirements as Two RIL populations were developed by crossing PI 567690
qSW_Gm standing for qtl_Slow Wilting_Glycine max_chromosome number. to Pana and PI 567731 to Magellan (Supplementary Fig. S1).
646 | Ye et al.

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Fig. 2.  Differences in transpiration rates of the parental lines under increasing vapor pressure deficit (VPD) and silver nitrate treatments. (A) Transpiration
rates of the parental lines under increasing VPD. The transpiration rates were calculated according to Equation 2. X0 is the breakpoint between the
two-line segments and R2 represents the portion of variations explained by the model. (B) Responses of transpiration rates of the parental lines to an
aquaporin inhibitor. AgNO3 was applied to the detached shoots (4 weeks after germination) and the percentage reduction in transpiration rate for each
individual plant due to the inhibitor treatment was calculated using Equation 3. At 4 weeks after emergence, the transpiration response of detached
shoots fed with silver ions was measured. Columns and bars represent the mean and SDs of 10 replicates. Duncan multiple comparison was performed
to compare the means and categorize the data into ‘a,’ ‘b,’ and ‘c’ at P<0.001.

WSs were rated for these RIL populations at three locations suggesting WS as a quantitative trait in these populations and
for 1–3  years (Fig. 1A). The WSs of the two RIL popula- no need for data normalization. Transgressive segregations in
tions were quantitatively and normally distributed (Fig. 3), WSs were observed in both mapping populations for all the
 Slow canopy wilting improves drought tolerance in soybean  |  647

three locations (Fig. 3). These results indicated that both elite associated with canopy WSs from each year and location
and exotic parents should have a donor locus or loci for SW. (Table 2). From the Pana×PI 567690 population, eight QTLs
Broad-sense heritability was detected as 0.42 and 0.47 for the were identified to locate at similar chromosomal positions to
Pana×PI 567690 and Magellan×PI 567731 populations, re- those previously reported by Abdel-Haleem et al. (2012) and
spectively, across all environments (years and locations) (Fig. 3). Hwang et al. (2015) (Table 2). From the Magellan×PI 567731
The evaluation of a possible relationship between canopy population, two new QTLs, named as qSW_Gm06 and qSW_
wilting and yield was carried out using 46 RILs from the two Gm10, were mapped as being distinct from those previously
populations. These RILs were selected for extreme perform- reported (Table 2).
ance in WS with <1.5 (23 SW RILs) and >3 (23 FW RILs) Among the 10 QTLs from the present mapping work, six
(Supplementary Table S1). These 46 RILs have similar agro- were identified from two to three environments (either dif-
nomic traits, including maturity, plant height, lodging (low), ferent year or different location) (Table 2; Fig. 5). Four of them
and determinant growth habit (Supplementary Table S1). were consistently mapped in two environments with favorable
Under non-stress (irrigated) conditions, the FW RILs showed a alleles from PI 567690 for SW, with each explaining 7.8% to
yield advantage over the SW RILs of 7.8–10.3% at both tested 10.4% of total phenotypic variations (Fig. 5). The other two

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locations; however, these differences were not statistically sig- novel QTLs (qSW_Gm06 and qSW_Gm10) were mapped in
nificant (Fig. 4). Under drought stress (rain-fed) conditions, the the Magellan×PI 567731 cross in three environments (Fig. 5).
FW RILs showed yield reductions of 48.5% and 36.8% at the Magellan and PI 567731 were found to contribute donor al-
two locations, while the SW RILs showed much lower yield leles for SW at qSW_Gm06 and qSW_Gm10, respectively
reductions of 35.4% and 22.9%, respectively (Fig. 4). The SW (Fig. 5). Moreover, qSW_Gm06 and qSW_Gm10 were identi-
RILs showed statistically significant yield advantages of 12.8% fied to have relatively large contributions to SW phenotypes,
and 13.7% over the FW lines under water-limited conditions with each explaining 20–29.6% of total phenotypic variations
at both locations (Fig. 4). When yield of the selected RILs was (Fig.  5). It was noted that among the six QTLs from mul-
evaluated separately for each population, a trend similar to the tiple environments, four QTLs (qSW_Gm05, qSW_Gm06,
combination of the RILs from the two populations was ob- qSW_Gm09, and qSW_Gm10) were detected at both hill
served (Supplementary Tables S2, S3); however, the difference plots (BREC or DC) and row plots (SA), while the other two
was not significant between FW and SW RILs under either (qSW_Gm12 and qSW_Gm19) were detected only at hill plots
condition due to reduced sample size. (Fig. 5). Other confounding factors, such as plant density, could
result in the difference in QTL detection between hill plots
QTLs for SW under drought in the elite×exotic crosses and row plots.

Linkage maps of the two RIL populations were constructed Isolation and confirmation of the major QTLs qSW_
using 1601 and 2516 polymorphic SNP markers acquired from Gm06 and qSW_Gm10
BARCSoySNP6K Illumina Infinium BeadChips genotyping
with an average genetic interval of 1.5 cM (Table 1). Subsequent Previously, none of the SW QTLs was confirmed in the same
QTL analyses in the two RIL populations identified 10 QTLs genetic background, such as a near-isogenic background. As

Pana × PI 567690 Magellan × PI 567731

100 100
H2 = 0.42 Salina Salina
BREC BREC
Number of plants

80 DC 80 DC

60 60 H2 = 0.47

40 40

20 20

0 0
1 2 3 4 5 1 2 3 4 5
Canopy wilting score
Fig. 3.  Phenotypic distributions of canopy wilting scores (WSs) of the two recombinant inbred line mapping populations. The means of WSs from
multiple years for each location were used to calculate the phenotypic distributions. The arrows with solid lines or dashed lines point to the performance
of the exotic or cultivated parents, respectively. The performance of canopy wilting was averaged for all environments (locations and years) for the
parental lines. Broad-sense heritability (H2) was calculated for WSs across all environments (locations and years) using Equation 1.
648 | Ye et al.

Salina Delta Center

3.5 3.5
Slow wilting type Slow wilting type
P = 0.077 Fast wilting type Fast wilting type
3 10.3% 3

Yield (t/ha)
P = 0.055
2.5 2.5 7.8%
P = 0.018 P = 0.009
2 2 12.8%
13.7%
1.5 1.5

1 1
Irrigated Rain-fed Irrigated Rain-fed

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Fig. 4.  Roles of the canopy wilting trait in yield protection under drought. The two sets of RILs were planted in the field under irrigated and rain-fed
conditions at Salina (left) and Delta Center (right). Data shown are means ±SDs of yield plots of 23 RILs for either slow wilting or fast wilting types.
Student’s t-test was performed to compare means of traits between the two sets of RILs. The percentages on the columns show the yield advantage of
the corresponding wilting types over the others.

Table 1.  Information of the constructed linkage maps for the two recombinant inbred line populations

Pana×PI 567690 Magellan×PI 567731

Chr. No. of markers Average interval (cM) No. of markers Average interval (cM)
1 82 1.1 144 1.6
2 74 1.9 147 1.0
3 89 1.6 114 1.9
4 95 1.2 130 1.5
5 54 2.2 102 1.7
6 87 1.3 126 1.6
7 56 2.1 144 1.5
8 134 2.1 221 1.1
9 80 1.3 130 1.5
10 81 1.2 91 2.0
11 63 1.4 73 2.6
12 62 1.3 110 1.6
13 96 1.4 179 1.1
14 66 1.4 95 1.8
15 69 1.6 113 1.8
16 68 1.4 107 1.7
17 83 1.6 106 1.7
18 85 1.3 151 1.3
19 56 1.6 93 1.4
20 121 1.8 140 1.3
Total 1601 1.5 2516 1.5

the two major SW QTLs (qSW_Gm06 and qSW_Gm10) were (Supplementary Fig. S1). ‘aa’ or ‘AA’ stands for the NILs with
identified in the present study, efforts were made to isolate and the donor allele for FW or SW at qSW_Gm06; while ‘bb’ or
confirm these two QTLs at a near-isogenic background. One ‘BB’ stands for the NILs with the donor allele for FW or SW at
RIL (RIL#049) in the F7:8 generation was identified to be het- qSW_Gm10. Line aabb showed an obvious wilting phenotype
erozygous at the both qSW_Gm06 and qSW_Gm10 regions by under drought (3 weeks without rainfall) (Fig. 6A). Adding
genotyping of the QTL flanking markers: Gm06_43235059/ SW alleles of either QTL significantly suppressed the canopy
Gm06_47338142 and Gm10_43894668/Gm10_44744804, wilting from score 3.3 to <1.9 (Fig. 2B). The line AABB has
respectively (Supplementary Fig. S1). The progenies of the lightest appearance of canopy wilting; however, the dif-
RIL#049 with homozygous genotypes at the two loci were ferences among AAbb, aaBB, and AABB are not statistically
selected as NILs for qSW_Gm06 and qSW_Gm10. These significant, possibly due to small sample sizes and inadequate
four selected NILs were named aabb, AAbb, aaBB, and AABB drought stress or the effects of undetected minor loci that are
 Slow canopy wilting improves drought tolerance in soybean  |  649

Table 2.  Summary of QTLs detected in previous and present genetic mapping

QTL Donor line Abdel-Haleem et al. (2012) Hwang et al. (2015) This research
qSW_Gm02-1 Benning, Jackson, PI 567690 – 2 populationsa 1 environmenta
qSW_Gm02-2 A5959, PI 416937, PI 567690 2 environmentsa 1 population 1 environment
qSW_Gm02-3 PI 416937 – 2 populations –
qSW_Gm04 PI 416937, PI 567690 2 environments – 1 environment
qSW_Gm05 PI 416937, Jackson, PI 567690 2 environments 1 population 2 environment
qSW_Gm06 Magellan – – 3 environments
qSW_Gm08 Jackson, Nannong 1138-2 – 2 populations –
qSW_Gm09 Jackson, A5959, PI 567690 – 2 populations 2 environments
qSW_Gm10 PI 567731 – – 3 environments
qSW_Gm11 PI 416937, Jackson, PI 424140 – 3 populations –
qSW_Gm12 PI 416937, PI 567690 5 environments – 2 environments

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qSW_Gm14 Jackson, PI 416937 – 2 populations –
qSW_Gm17-1 KS4895, A5959, Benning, Pana 2 environments 4 populations 1 environment
QSW_Gm17-2 KS4895 – 2 populations –
qSW_Gm19 PI 416937, Jackson, PI 424140, PI 567690 2 environments 3 populations 2 environments

a
Numbers of environments or populations in which the QTL was detected in the corresponding study.

Gm05 Gm06 Gm09

PI 567690
R2: 10.4%
Gm05_2548644
2014_SA PI 567690
2015_DC R2: 8.0%
Gm09_36869408
Magellan 2014_SA
R2: 29.6% 2014_DC
Gm06_43432590
2013_SA
2015_SA
2015_BREC

Gm10 Gm12 Gm19

PI 567690
R2: 9.3%
Gm19_45553057
PI 567731 PI 567690 2014_DC
R2: 20.0% R2: 7.8% 2015_DC
Gm10_44445941 Gm12_37803362
2013_SA 2014_BREC
2015_SA 2015_BREC
2015_BREC

Fig. 5.  Chromosomal locations and genetic components of the two QTLs associated with canopy wilting score (WS) detected at multiple environments.
The linkage maps were constructed based on genotyping results from Illumina 6K SNP arrays using JoinMap 4.0 (Table 1). The annotations of each
QTL show the lines having the donor alleles for the slow wilting trait, the largest percentage contributions of the QTL to the total phenotypic variations at
multiple environments, the nearest markers of the QTL, and environments detecting the QTL, respectively. Only QTLs detected in ≥2 environments are
shown.
650 | Ye et al.

A B
4.5
aabb AAbb 4

Canopy wilting score

3.5 c
3
2.5
d d
aaBB AABB 2 d
1.5
1

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0.5
aabb AAbb aaBB AABB
Fig. 6.  Genotypic differences in canopy wilting under drought of qSW_Gm06 and 10. (A) Representative images of canopy wilting phenotypes of the
near-isogenic lines (NILs) for qSW_Gm06 and 10. Images were taken at noon after 3 weeks without rainfall. ‘aa’ or ‘AA’ stand for the NILs with the donor
allele for fast wilting or slow wilting at qSW_Gm06, while ‘bb’ or ‘BB’ stand for the NILs with the donor allele for fast wilting or slow wilting at qSW_Gm10.
(B) Canopy wilting scores of the NILs. Bars represent means and SDs of 9–11 plants for each genotype. The letters ‘c’ or ‘d’ indicate results from
Duncan’s multiple comparison test for not significant (same letter) or significant (different letter) at P<0.002.

still segregating in the NILs (Fig. 6B).Yield test of these NILs is these lines was shown to be sensitive to the AgNO3 treatment
needed to confirm the yield benefit of these two QTLs under and the three BP lines even showed greater sensitivity than the
well-controlled drought in future studies. These efforts suc- non-BP line (Fig. 2B). These results indicate that the mech-
cessfully isolated the two novel QTLs from a complex genetic anism for the limited maximum transpiration in SW genotypes
background into a near-isogenic background using marker- could be independent of the previously hypothesized lack of
assisted selection (MAS) and confirmed the effects of the SW silver-sensitive aquaporins.
QTLs for the first time. Previously, PI 416937 was reported to have a much larger
root system in the field than other FW lines, indicating the ex-
istence of other mechanisms enhancing the SW phenotype in
Discussion addition to water conservation ability through limited transpir-
Physiological mechanisms of SW ation responses (Hudak and Patterson, 1995). Therefore, other
mechanisms, such as a larger and/or deeper root system, could
The SW phenotype is a complex trait that could be involved also be present in the new SW lines in this research, which
in water conservation strategies in leaves and water resource have not been identified yet. Dissecting the SW traits into indi-
exploration by roots (Valliyodan et  al., 2017). Modification vidual genetic loci is needed to allow more in-depth mechan-
in transpiration was considered one important strategy for istic studies for a full understanding of this complex trait. The
plants to adapt to water-limited conditions. Transpiration of development of NILs for SW QTLs enabled zooming in on
plants increases with increasing atmospheric VPD, but, in some specific loci/genes and conducting more extensive mechan-
genotypes, the TR becomes limited when a certain VPD is istic studies towards better understanding of physiological and
reached (Sinclair and Bennett, 1998). This limited transpir- molecular mechanisms for SW.
ation response may be important in conserving soil water for
water deficit conditions. A visual consequence of the limited Impact of SW on yield
transpiration response can be delayed wilting (i.e. SW) under
drought conditions (Fletcher et al., 2007). Previously, lack of a The SW trait was considered to be especially desirable in en-
specific silver-sensitive population of aquaporins was hypothe- vironments of low humidity/high VPD for water conservation
sized to explain the limited maximum TR of PI 416937 under (Fletcher et al., 2007). In a simulation analysis, the imposition
increasing VPD (Sadok and Sinclair, 2010). In the present study, of the trait of limited maximum TRs was predicted to improve
three lines (PI 567690, PI 567731, and Magellan) showed BPs yield under drought by >75% (Sinclair et al., 2010). Previous
in TR at a VPD of 1.38–2.00 kPa (Fig. 2A). PI 567690 was analysis of soybean production in the USA indicated that those
particularly interesting, because the slope above the BP was genotypes expressing the limited maximum TRs were likely
essentially horizontal, similar to that found in PI 416937 (Fig. to yield more due to significant water savings under non-
2A), suggesting that this PI should have stronger suppression irrigated and high VPD conditions (Sinclair et al., 2010). In the
in transpiration under drought. These four parental lines were present study, we found that RILs with the SW trait showed a
subjected to AgNO3 treatment to determine if this test could significantly greater yield under non-irrigated and high VPD
confirm the previous hypothesis with PI 416937. However, conditions as compared with RILs with the FW phenotype
unlike the findings in PI 416937, the transpiration of all of (Fig. 4). These results offered experimental evidence for the
 Slow canopy wilting improves drought tolerance in soybean  |  651

first time to support the previous hypothesis that the SW trait QTLs in different genetic backgrounds (Table 2). More inter-
is associated with drought tolerance in soybean. estingly, two new SW QTLs (qSW_Gm06 and qSW_Gm10)
However, when water supply is not limiting, a limiting max- were mapped in the Magellan×PI 567731 population and each
imum TR may result in yield penalty (Sinclair et al., 2005). PI parent contributes to SW at one locus (Fig. 5). Although these
416937 was found to have a low yield potential (Cho et  al., two QTLs have relatively larger effects on SW and are more
2003). The low yield of PI 416937 was thought to be con- stable across different environments compared with the others
sistent with a limiting maximum TR (Fletcher et  al., 2007). (Fig. 5), further confirmation of these QTLs in a similar gen-
However, the low yield of this PI line could also be due to its etic background is still necessary. Therefore, these two QTLs
exotic genetic background, as the exotic germplasms tend to were isolated into a near-isogenic background by MAS in the
have much lower yield potentials compared with cultivars. In heterogeneous inbred families (see Supplementary Fig. S1). As
the present study, we tested the yield potentials of both SW expected, preliminary field evaluations of the NILs successfully
and FW RILs and found that the SW trait had yield penalties confirmed these two QTLs for the SW phenotype (Fig. 6).
of 7.8–10.3% in irrigated environments, where water supply The confirmation of the two SW QTLs in the NILs developed
is sufficient (Fig. 4). In this experiment, the 46 tested RILs by MAS indicates that it is feasible and recommended to use

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were selected from the same RIL populations to normalize MAS for this complex trait in soybean breeding, especially for
the genetic backgrounds between SW and FW genotypes. the major QTLs, such as qSW_Gm06 and qSW_Gm10.
Therefore, it can be concluded that a limiting maximum TR It was hypothesized that stacking of SW QTLs could im-
increases yield stability, but with associated yield penalty in op- prove drought tolerance significantly, especially under severe
timal growth seasons or areas. These results can be interpreted or extended drought periods (Valliyodan et al., 2017). In this
as suggesting that the SW phenotype is particularly useful for research, stacking both QTLs showed a slight enhancement in
breeding soybean varieties for environments with high VPD SW performance compared with adding either QTL, although
and low water supply. the difference is statistically insignificant (Fig. 6).This insignifi-
cance could be due to small sample sizes or inadequate drought
Quantitative inheritance and complexity of the SW trait stress (only 3 weeks without rainfall). Further evaluations of
the benefit (canopy wilting and yield) of stacking SW QTLs
Previously, >10 SW QTLs were mapped from several popu- are needed with a larger sample size under better controlled
lations and/or environments. The majority of these QTLs drought conditions.
(11/13) were contributed from PI 416937 and Jackson, while
the FW parents still offered donor alleles at a few loci (Table
2). However, most of the mapped QTLs were found to be un- Supplementary data
stable across environments. For instance, the major QTL on
Chr. 12, previously reported by Abdel-Haleem et  al. (2012), Supplementary data are available at JXB online.
was not detectable in the same population reported by Hwang Fig. S1. A scheme to develop recombinant inbred line (RIL)
et al. (2015, 2016). GWAS in the diverse panel also confirmed mapping populations and near-isogenic lines (NILs) for qSW_
the complexity of SW, as 23 putative loci had a statistically sig- Gm06 and qSW_Gm10.
nificant association with this trait (Kaler et al., 2017). However, Table S1. Recombinant inbred lines selected for yield test.
at this point, none of these QTLs was reported to be con- Table S2. Yield of the selected contrasting recombinant in-
firmed at a synchronized genetic background, such as a near- bred lines from the Pana×PI 567690 population under irri-
isogenic background. gated and rain-fed conditions.
In this study, six QTLs were mapped in two RIL populations Table S3. Yield of the selected contrasting recombinant in-
under more than two environments (Table 2; Fig. 5). The ma- bred lines from the Magellan×PI 567731 population under ir-
jority of the QTLs (four out of six) were detected at both hill rigated and rain-fed conditions.
plots and row plots (Fig. 5), suggesting that hill plots can also be
used to evaluate the canopy wilting trait. However, two QTLs
were detected only in hill plots (Fig. 5), suggesting the exist- Acknowledgements
ence of other confounding factors in hill plot planting, such as The authors gratefully acknowledge the financial support for this study
plant density. The difference in plant density can affect avail- provided by the United Soybean Board and USDA-NIFA (1006057).The
able water resources in the soil and may ultimately affect the authors acknowledge Mackensie Murphy and Dennis Yungbluth, Division
development of canopy wilting phenotypes. In future studies, of Plant Sciences, the University of Missouri, for their technical assistance.
row plots are recommended to conduct canopy wilting and
drought tolerance evaluation, if space and resources are avail-
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